A protective vaccine against HIV-1 is widely believed to require, in part, induction of a potent neutralizing humoral response, but current vaccine candidates do not approach this goal, nor are the epitopes that might mediate such neutralization known. Although the sequence diversity in the V3 loop is generally believed to make this domain unsuitable as a vaccine target, our recent results suggest that there are conserved epitopes within V3 that mediate potent cross-neutralization of primary viruses within and across clades. These results were obtained using a novel V3 antigen, produced in mammalian cells as a fusion protein, that is properly glycosylated and at least ten times more reactive with HIV-1+ human patient sera than are synthetic peptides. Fusion proteins expressing clade B and clade A V3 domains have been used to isolate polyclonal V3-reactive antibody fractions from human sera from patients infected with clade B or clade A viruses and to screen new V3-reactive human monoclonal antibodies (mabs) with a number of different patterns of cross-neutralization activity against primary isolates. This proposal seeks to further characterize such polyclonal and monoclonal V3-reactive antibodies in order to better define the epitopes that mediate cross-neutralization. Polyclonal V3-reactive neutralizing antibodies will be analyzed by determining the ability of various antigens to adsorb out the neutralizing activity. These antigens will include synthetic peptides, de-glycosylated V3 fusion proteins, fusion proteins expressing linearized V3 domains, and fusion proteins expressing V3 domains with alanine substitutions at different locations. V3-reactive mabs will be characterized by ELISA against these antigens, against soluble gp120, and against Env complexes on the viral surface. We will attempt to exploit our finding that neutralization sensitivity to V3-directed antibodies is regulated by the Vl/V2 domain to generate chimeric Envs suitable for extending studies on the breadth of cross-neutralization mediated by polyclonal V3-reactive antibody fractions in which the concentration of relevant antibodies may be low. We will also isolate and characterize new V3-reactive mabs using transgenic mice that express fully human lgGs (XenoMouse, Abgenix). We will also determine whether isolated V3 domains expressed on fusion proteins and carrying a synthetic TH epitope are able to induce high titer neutralizing antibodies against primary viruses. These studies will increase our understanding of conserved neutralizing epitopes in V3, and may lead directly to a vaccine candidate for further development.